Human-derived tumor cell growth inhibitors

ABSTRACT

A DNA fragment encoding a novel human-derived tumor cell growth inhibitor can be obtained from a cDNA library originating from human colon tumor cell, using as a DNA probe a DNA fragment encoding mouse human-derived tumor cell growth inhibitor. An expression plasmid bearing a promoter originating from Bacillus brevis, a signal peptide originating from Bacillus brevis and a DNA fragment encoding human-derived tumor cell growth inhibitor downstream of the signal peptide is constructed and Bacillus brevis is transformed by the expression plasmid. The resulting transformant is incubated to secret the novel inhibitor out of the cell so that the novel inhibitor can be produced efficiently.

This application is a PCT/JP94/100895 filed Jun. 2, 1994.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a novel human-derived tumor cell growthinhibitor, a DNA fragment encoding the inhibitor, an expression plasmidof said human-derived tumor cell growth inhibitor bearing the DNAfragment, Bacillus brevis transformed by the expression plasmid, and amethod for producing the human-derived tumor cell growth inhibitor usingBacillus brevis by a genetic engineering technique.

2. Background Art

Synthetic drugs such as chemotherapeutic agents or immunotherapeuticagents have been heretofore widely used as anti-tumor agents. However,these drugs generally encounter problems that their specificity is lowand side-effects are serious. On the other hand, many tumor cell growthinhibitors have been found in tissue culture cells. These inhibitorscould be such anti-tumor agents that would be highly specific and wouldhave minimized side-effects. Representative examples of such inhibitorsare interferon, lymphotoxin and tumor necrosis factor (TNF).

Recently, a tumor cell cytotoxic factor obtained from human fibroblastand a tumor cell growth inhibitor obtained from human lung cancer cellsare reported in Japanese Patent KOKAI Nos. 1-148197 and 1-187094,respectively.

On the other hand, some cell growth inhibitors are isolated from thefibroblastic 3T3 cell line established from the cells obtained fromSwiss fetal mice. For example, Natraj et al. has reported that a growthinhibitor was obtained from the cell membrane of 3T3 cells in thestationary phase, cf., Proc. Natl. Acad. Sci. U.S.A., 75, 6115-6119(1978). Harel et al. has reported that a growth inhibitor having amolecular weight of 40 kDa was obtained from the culture supernatant of3T3 cells, see J. Cell. Physiol., 119, 101-106 (1984), ibid., 123,139-143 (1985). However, it is known that these growth inhibitors allfail to show any significant inhibitory action on tumor cells.

A mouse-derived tumor cell growth inhibitor isolated and purified fromthe culture supernatant of the established cell line NIH3T3-sf, which isobtained by focus cloning from NIH3T3 cells (J. Virol., 4, 549 (1969)),one of fibroblastic 3T3 cell lines established from Swiss fetal mice, isreported in Japanese Patent Application No. 3-11950 as the inhibitorhaving a significantly inhibitory activity on tumor cells. Thismouse-derived tumor cell growth inhibitor exhibits a potent growthinhibition activity on tumor cells such as human promyelogenous leukemiacells or human cervical carcionoma cells and is expected to be effectiveas a new drug for the treatment of cancer. However, the inhibitor is amouse-derived one so that there might be an antigenicity problem whenapplied to human.

DISCLOSURE OF THE INVENTION

The present inventors previously succeeded in cloning of,cDNA encodingthe mouse-derived tumor cell growth inhibitor which was isolated fromthe culture supernatant of NIH3T3-sf cells described above and alreadyfiled PCT/JP92/01580 directed to its nucleotide sequence.

In order to obtain a novel human-derived tumor cell growth inhibitor,the present inventors have made studies on cloning of cDNA encoding ahuman-derived tumor cell growth inhibitor from human placentachromosomal DNA-derived DNA library and from human colon tumorcell-derived cDNA library and found a novel human-derived tumor cellgrowth inhibitor and a DNA fragment encoding the inhibitor.

The present inventors have made further studies to provide anindustrially advantageous method for producing the inhibitor byrecombinant DNA technique, using the DNA fragment encoding thehuman-derived tumor cell growth inhibitor. As a result, it has beendiscovered that by expressing the inhibitor using a promoter and a DNAfragment encoding a signal perptide as a regulator gene, derived fromBacillus brevis and using Bacillus brevis as a host, the inhibitor canbe secreted out of the cell to produce the inhibitor in a largequantity. The present invention has thus been accomplished.

That is, an object of the present invention is to provide ahuman-derived tumor cell growth inhibitor having an amino acid sequencerepresented by formula (1):(Seq. I.D. No. 1) ##STR1##

Another object of the present invention is to provide a DNA fragmentencoding the human-derived tumor cell growth inhibitor having the aminoacid sequence shown by formula (1).

A further object of the present invention is to provide a DNA sequencecomprising a promotor derived from Bacillus brevis, a DNA fragmentencoding a signal peptide derived from Bacillus brevis, and a DNAfragment coding for the human-derived tumor cell growth inhibitor havingthe amino acid sequence of formula (1) defined above.

A still further object of the present invention is to provide anexpression plasmid for the human-derived tumor cell growth inhibitorcomprising the DNA sequence described above.

A still further object of the present invention is to provide Bacillusbrevis transformed by the expression plasmid.

A still further object of the present invention is to provide a methodfor preparing the human-derived tumor cell growth inhibitor whichcomprises culturing Bacillus brevis described above in a medium toexpress and extracellularly secrete the human-derived tumor cell growthinhibitor, and then recovering the human-derived tumor cell growthinhibitor from the medium.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a nucleotide sequence of the DNA fragment encodingmouse-derived tumor cell growth inhibitor and its amino acid sequence.

FIG. 2 shows an analysis pattern, i.e., an autoradiogram of humanplacenta chromosomal DNA fragment by Southern hybridization.

FIG. 3 (a) shows an outline of the chromosomal DNA fragment encoding apart of the human-derived tumor cell growth inhibitor; (b) is acomparison in nucleotide sequence between a part of the human-derivedtumor cell growth inhibitor and a part of the cDNA fragment of amouse-derived tumor cell growth inhibitor precursor; and (c) is acomparison in amino acid sequence between a part of the human-derivedtumor cell growth inhibitor and a part of the cDNA fragment of themouse-derived tumor cell growth inhibitor precursor.

FIG. 4 indicates a nucleotide sequence of the cDNA fragment obtainedfrom human colonel tumor cell HCT-15 and an amino acid sequence deducedtherefrom.

FIG. 5 is a comparison in amino acid sequence between the human-derivedtumor cell growth inhibitor.

FIG. 6 shows a nucleotide sequence of the cDNA fragment encoding thehuman-derived tumor cell growth inhibitor and an amino acid sequencededuced therefrom.

FIG. 7 shows oligonucleotides used as primers for amplifying the DNAfragment by PCR.

FIG. 8 shows the structure of plasmid PBR-AN bearing NcoI site and a DNAsequence encoding a part of the C-terminal region of the signal peptideof MWP which is one of the major cell wall proteins of Bacillus brevis47.

FIG. 9 indicates construction of plasmid pKN120 used as a cloning vectorof the human-derived tumor cell growth inhibitor gene.

FIG. 10 shows digestion of plasmid pKN120 and the human-derived tumorcell growth inhibitor gene.

FIG. 10 shows digestion of plasmid pKN120 and the human-derived tumorcell growth inhibitor gene.

FIG. 11 shows construction of expression plasmid pBB230 of thehuman-derived tumor cell growth inhibitor.

FIG. 12 is a graph showing an elution profile of C18 reversed phase HPLCof the human-derived tumor cell growth inhibitor which is the subject ofthe present invention.

FIG. 13 is a graph showing a growth inhibitory activity of thehuman-derived tumor cell growth inhibitor on human tumor cells.

FIG. 14 is a photograph showing the results of Western blotting of thehuman-derived tumor cell growth inhibitor.

BEST MODE FOR CARRYING OUT THE INVENTION

The human-derived tumor cell growth inhibitor (abbreviated as hTG-700 orhuman TG-700) of the present invention can be obtained by cloning a DNAfragment encoding the inhibitor, constructing an expression vectorbearing the DNA fragment, inserting into a host cell for expression.Alternatively, the inhibitor can also be obtained by chemical synthesisbased on the amino acid sequence deduced from the DNA fragment encodinghTG-700.

The DNA fragment encoding hTG-700 can be prepared by cloning from humancell-derived CDNA library using mouse-derived tumor cell growthinhibitor (hereinafter abbreviated as mTG-700 or mosue TG-700) having aknown amino acid sequence.

mTG-700 is isolated from the culture supernatant of NIH3T3-sf, which isestablished cell line derived from 3T3 cell and its amino acid sequenceis reported in Japanese Patent Application Laid-Open No. 4-211698 and EP460910. The amino acid sequence of mTG-700 is shown in FIG. 1. Cloningof cDNA encoding the amino acid sequence can be performed in aconventional manner.

Firstly, mRNA is isolated and purified from NIH3T3-sf cells, cDNAcomplementary of mRNA is prepared and bound to lambda phage gtlO . Phageparticles are then formed by in vitro packaging (Hohn et al., Proc.Natl. Acad. Sci. U.S.A., 74, 3259 (1977)) to obtain cDNA library. Next,some oligonucleotides deduced from the amino acid sequence of mTG-700are chemically synthesized. Using the oligonucleotides as primers andthe cDNA library above as a template, various DNA fragments encodingmTG-700 are amplified by the PCR method (Saiki et al., Science, 230,1350 (1985)). These nucleotide sequences are determined by the dideoxychain terminator method (Sanger, F. et al., Proc. Natl. Acad. Sci.U.S.A., 74, 5463 (1977)). Based on the information and the amino acidsequence of mTG-700, the nucleotide sequence of DNA encoding mTG-700 canbe determined. The thus determined nucleotide sequence of the DNAfragment encoding mTG-700 is shown in FIG. 1.

Using the DNA fragment encoding mTG-700 as a probe, cDNA encodinghTG-700 can be cloned from human-derived cDNA library, e.g., humanplacenta chromosomal DNA library or human colon tumor cell-derived cDNAlibrary.

More specifically, the cloning of cDNA encoding hTG-700 can be performedby the following procedure.

First, DNA fragment encoding mTG-700 having the nucleotide sequenceshown in FIG. 1 is labeled with, e.g., α-³² p! dCTP by multi-primed DNAlabeling system (Amersham Co., Ltd.) to prepare DNA probe. Using the soprepared probe, human placenta chromosomal DNA (Clonetech Co., Ltd.) isdigested with an appropriate restriction enzyme, e.g., HaeIII; DNAfragments obtained are subjected to Southern hybridization. DNA fragmentconsidered to contain a gene highly homologous to the DNA fragmentcapable of hybridizing the probe and encoding mTG-700 can be detected bySouthern hybridization and its molecular weight can be deduced. Then,DNA fragment around the deduced molecular weight is extracted from,e.g., agarose gel.

This DNA fragment is mixed with, e.g., lambda phage gtlO-EcoRI arm tobind to each other, using T4 DNA ligase. A vector is thus constructed.Next, phage particles are formed by the in vitro packaging method toobtain human placenta chromosomal DNA fragment library.

With regard to the library, hybridization is performed using the sameprobe as described above, namely, the DNA fragment encoding mTG-700labeled with a-³² p! dCTP. The following screening gives a cloneconsidered to bear a gene highly homologous to the DNA fragment encodingmTG-700. DNA is extracted from the clone and the nucleotide sequence ofthe DNA fragment extracted is determined by the dideoxy chain terminatormethod. FIG. 3 shows the nucleotide sequence of the DNA fragment thusdetermined. As is noted from FIG. 3, among the human placentachromosomal DNA fragments obtained by digestion with restriction enzymeHaeIII, a DNA fragment having an amino acid showing high homology tomTG-700 is contained in the nucleotide sequence from 552 to 675 and isconsidered to be hTG-700.

Next, PstI fragment (520-674 nucleotide sequence in FIG. 3) in the humanplacenta chromosomal DNA fragment is labeled with a-³² p! dCTP toprepare DNA probe. Using this DNA probe, cDNA is synthesized from MRNAof human colon tumor cell HCT-15 (Dainippon Pharmaceutical Co., Ltd.,Cancer Research, 39, 1020 (1979)) and mixed with lambda phage gtlO-EcoRIarm. The cDNA library prepared by the in vitro packaging method is thensubjected to screening.

A clone that can hybridize the DNA probe is obtained and DNA isextracted from the clone. The nucleotide sequence of DNA is determinedby the dideoxy chain terminator method and compared with the nucleotidesequence of the DNA fragment encoding mTG-700. Thus, the nucleotidesequence of the desired DNA fragment encoding hTG-700 can be determined.

The nucleotide sequence of the DNA fragment encoding hTG-700 thusdetermined is shown by formula (2) (Seq. I.D. No. 2) below: ##STR2##

The DNA fragment encoding hTG-700 having the nucleotide sequence offormula (2) determined as described above may also be chemicallysynthesized by known methods, e.g., by the triester phosphate method(Letsinger et al., J. Am. Chem. Soc., 91, 3350 (1969)).

Alternatively, the DNA fragment encoding hTG-700 may be prepared in alarge quantity by synthesizing oligonucleotides corresponding to the5'terminus and 3'terminus of the DNA fragment encoding hTG-700 using asa template the cDNA library prepared from mRNA of HCT-15 cells describedabove and amplifying the DNA fragment by PCR (Saiki et al., Science,230, 1350 (1985)).

Based on the nucleotide sequence of the DNA fragment encoding hTG-700thus cloned, the amino acid sequence of hTG-700 is determined and shownby formula (1) (Seq. I.D. No. 1) below: ##STR3##

The hTG-700 having the amino acid sequence of formula (1) can bechemically synthesized by, e.g., the solid phase method described inMerrifield, J. Am. Chem. Soc., 85, 2185 (1963)). In general, chemicalsynthesis based on the solid phase method may be carried out using apeptide automatic synthesizer, by following the standard operationprogram.

The hTG-700 of the present invention may also be prepared by recombinantDNA technique using the DNA fragment encoding hTG-700. That is, hTG-700can be expressed using the promoter originating from Bacillus brevis andthe DNA fragment encoding the signal peptide as a regulator gene andusing Bacillus brevis as a host to secrete a large quantity of hTG-700out of the cell. It is thus possible to produce hTG-700 in a largequantity.

In these years, a keen attention has been increasingly brought toBacillus brevis as a host used for recombinant DNA technique, since thebacteria hardly produces extracellular protease, see Udaka, Journal ofJapan Agrichemical Association, 61 (6), 669-676 (1987). Typical examplesof the promoter originating from Bacillus brevis include promoters forgenes of major cell wall protein (MWP) of Bacillus brevis 47 (FERMP-7224) and Bacillus brevis H102 (FERM BP-1087). As the DNA fragmentencoding the signal peptide derived from Bacillus brevis, there may beused a DNA fragment coding for the signal peptide of MWP gene fromBacillus brevis 47 or from Bacillus brevis H102. Such a signalpeptide-coding DNA fragment is employed to secret hTG-700 out of thecell. In order to express hTG-700, SD sequence derived from Bacillusbrevis at the 3' terminus of the promoter described above, initiationcodon at the 3' terminus, the DNA fragment encoding the signal peptidedescribed above and the DNA fragment encoding hTG-700 are connected inthis order.

The promoter, the DNA fragment of SD sequence and the DNA fragmentencoding the signal peptide are known and described in S. Udaka et al.,Biotechnology and Genetic Engineering Reviews, 7, 113-146 (1989) andhence can be prepared by chemical synthesis through the phosphatetriester method above. These fragments may also be prepared by cloningfrom, e.g., Bacillus brevis 47. These DNA fragments may be ligated toeach other by utilizing an appropriate restriction enzyme site or via anappropriate linker. Alternatively, vectors such as pNU200 are known tobear the promoter, SD sequence and signal peptide-coding DNA fragmentfor the major cell wall protein gene from Bacillus brevis 47 (Udaka,Journal of Japan Agrichemical Association, 61 (6), 669-676 (1987) andUdaka et al., Proc. Natl. Acad. Sci. USA, 86, 3589-3593 (1989)) andaccordingly, these vectors can be employed in its original form, withoutany modification.

More specifically, the expression vector can be constructed, e.g., bythe following embodiment.

That is, the hTG-700 region is amplified by PCR, using as a templatecDNA library containing cloned cDNA encoding hTG-700, to prepareMWP-hTG-700; MWP-hTG-700 is a DNA fragment bearing the hTG-700-codingDNA fragment and the DNA fragment with a part of the signalpeptide-coding DNA sequence at the 5' terminus thereof and a terminationcodon at the 3' terminus, see FIG. 10.

On the other hand, plasmid pKN120 having a part of the DNA sequenceencoding the signal peptide and having the restriction enzyme NcoI sitefor ligation to the gene encoding hTG-700 is constructed as shown inFIG. 9.

Then MWP-hTG-700 is ligated to pKN120 by utilizing the restrictionenzyme site NcoI to construct plasmid 46hTG-RF, see FIG. 11. Thisplasmid contains a part of the DNA sequence encoding the signal peptideand the hTG-700-coding DNA fragment and a termination codon subsequentthereto.

Next, ApaLl-HindIII DNA fragment containing MWP-hTG-700 is excised fromthe plasmid 46hTG-RF as shown in FIG. 11.

On the other hand, plasmid pNU200 is treated with restriction enzyme toobtain a DNA fragment bearing the promoter derived from Bacilus brevis,SD sequence and the DNA sequence encoding a part of the amino acidsequence in the N-terminal domain of the signal peptide. This DNAfragment is ligated to the MWP-hTG-700-bearing DNA fragment describedabove so that the desired expression plasmid pBB230 can be constructed,see FIG. 11.

In addition to the methods described above, the expression plasmid mayalso be constructed in a similar manner except for using, e.g., knownplasmid pHY500. Plasmid pHY500 is known as a vector containing the DNAfragment encoding the promotor, SD sequence and signal peptide of themajor cell wall protein gene from Bacillus brevis 47, see H. Yamagata etal., Proc. Natl. Acad. Sci. USA, 86, 3589-3593 (1989).

Representative examples of the host used to express hTG-700 are Bacillusbrevis 47 , Bacillus brevis 47-5 (FERM BP-1664) and mutants thereof.

A transformant can be obtained by inserting the expression plasmid intoBacillus brevis in a known manner (Takahashi et al., J. Bacteriol., 156,1130 (1983).

The transformant is subjected to shake culture in an appropriate medium,e.g., T₃ modified medium (H. Yamagata et al., Proc. Natl. Acad. Sci.USA, 86, 3589-3593 (1989)) at a temperature of 15 to 42° C., preferablyat 24 to 37° C., Continuous culture for 16 to 166 hours, preferably for24 to 120 hours results in secretion of hTG-700 in the medium in such aform that the active steric structure is maintained.

The thus secreted hTG-700 can be purified from the medium in aconventional manner such as salting out, gel filtration chromatography,ion exchange chromatography, reversed phase chromatography, alone or incombination. The desired hTG-700 can thus be obtained. hTG-700 of thepresent invention may be in the form of pharmacologically acceptablesalt. Examples of such salts are acid addition salts, e.g.,hydrochloride, hydrobromide, hydroiodide, sulfate, acetate, citrate,succinate, fumarate, oxalate and p-toluenesulfonate; and base additionsalts such as potassium, sodium, calcium, aluminum and ammonium salts.

hTG-700 of the present invention is effective for the treatment ofleukemia, renal cancer and uterocervical cancer. Dosage of hTG-700varies depending upon condition but a daily dose is generally in therange of 0.001 to 10 mg for adult. The dosage may be given once ordivided into two to 4 times. hTG-700 is prepared into solid preparationssuch as tablets, pills, capsules or granules, or preparations such asinjection, liquid, emulsion or suppositories. These preparations areprepared in a conventional manner; if necessary and desired,conventional additives such as aids, stabilizers, emulsifiers ordiluents may also be added to the preparations.

Hereinafter the present invention will be described below in moredetail, by referring to the examples and experiments.

EXAMPLE 1 Isolation of DNA fragment encoding hTG-700 and determinationof its nucleotide sequence

1. Isolation of human chromosomal DNA fragment encoding a part ofhTG-700

1) Analysis of human chromosomal DNA fragment by Southern hybridization

Southern hybridication of human chromosomal DNA fragment was carried outusing as a probe cDNA fragment encoding mTG-700 having the nucleotidesequence shown in FIG. 1.

That is, 10 μg of human placenta chromosomal DNA (Clonetech Co., Ltd.)was digested with HaeIII (100 units) at 37° C. for 15 hours. Thedigestion produce was subjected to electrophoresis by 0.8% agarose geland then transferred onto a nylon membrane (Hibond-N, Amersham Inc.)according to the method of Southern et al. (Southern, E.M., J. MO1.Biol., 98, 503 (1975). The DNA fragment was fixed by heat treatment at80° C for 2 hours. The DNA fragment was fixed by heat treatment at 80°C. for 2 hours. The filter on which the obtained DNA fragment wasimmersed in a solution for hybridization (6×SSC, 5×Denhardt's solution,0.5% SDS, 30% formamide, 100 μg/ml salmon sperm DNA fragment), followedby heating at 37° C. for 2 hours. Using multiprime DNA labeling system(Amersham Inc.), cDNA fragment encoding mTG-700 having the nucleotidesequence shown in FIG. 1 was labeled with α³² p! dCTP and used as aprobe. The probe was added to the solution for hybridization describedabove and the reaction was continued at 37° C. for 16 hours. After thereaction was completed, the filter was rinsed at room temperature with1×SSC buffer containing 0.1% SDS. After washing at 37° C. for further 30minutes with 0.1×SSC buffer containing 0.1% SDS, the filter wasair-dried. The filter was brought into close contact with X ray filmX-OMART AR (Eastman Kodak) and exposed to light at -80° C. for 16 hours.As the result, a band was observed at about 1200 base pairs, suggestingthat human gene considered to code for human-derived tumor cell growthinhibitor would be present, see FIG. 2.

2) Construction of human chromosomal DNA fragment

(1) Extraction of human chromosomal DNA fragment After 10 μg of humanplacenta chromosomal DNA (Clonetech) was digested with 100 units ofHaeIII in the manner described in 1) above, electrophoresis wasperformed one 0.8% agarose gel and the DNA fragment at about 1200 basepairs was extracted as follows.

a. The DNA fragment at about 1200 base pairs was adsorbed onto DE81Paper (Whatman) (Dretzen, G. et al., Anal. Biochem., 112, 295, 1981).

b. The paper was immersed in 300 μl of an eluent (1.5 M NaCl, 1 mM EDTA,10 mM Tris-HCL, pH 8.00.

c. A mixture of phenol and chloroform (1:1) was added in an amount of300 μl.

d. Agitation was followed by centrifugation at 10,000×g for 10 minutes.

e. The supernatant was withdrawn and 750 μl of ethanol was added to theresidue.

f. The mixture was allowed to stand at -70° C. for an hour.

g. The system was centrifuged at 10,000×g for 10 minutes.

h. After dissolving in 10 μl of TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH8.00), a solution containing the DNA fragment at about 1200 base pairswas obtained.

(2) Ligation of human chromosomal DNA fragment to vector

The DNA fragment obtained in (1) above was mixed with lambda phagegtl0-EcoRI arm (manufactured by Strategene Co., Ltd.) and the two wereligated to each other using T4 DNA ligase.

(3) In vitro packaging

Phage particles were produced from the vector-ligated DNA fragment usingin vitro packaging kit (Amersham) to construct the human chromosomal DNAlibrary.

3) Screening of the human chromosomal DNA fragment library

About 6×10⁵ of the recombinant phages obtained in 2)(3) described abovewere infected to 4 ml of E. coli NM514 which had been incubatedovernight at 37° C. in LB medium (trypton: 10 g, yeast extract: 5 g,sodium chloride: 10 g/L). The seed medium was then inoculated in 20 LBmedium plates of 9×14 cm each, containing 1.5% agar. Each of the LBmedium plates was overlaid by 3 ml of LB medium containing 0.75% agaroseand kept at 45° C. After incubation at 37° C. for 12 hours, a nylonmembrane filter (HIBOND-N; Amersham) was brought in close contact for aminute with the plate in which plaques were formed. The filter wasimmersed in an alkali solution (1.5 M NaCl, 0.5 N NaOH) for 2 minutesand then in a neutral solution (3.0 M NaCl, 0.5 N Tris-HCl pH 7.0) for 5minutes. After rinsing with 2×SSC, the filter was air-dried. Theresulting filter was treated in the same manner as in 1--1) describedabove to fix the DNA fragment. Hybridization was carried out using as aprobe the DNA fragment encoding mTG-700 labeled with α-³² p! dCTP, whichwas followed by exposure to light on a film. The area corresponding tothe sensitized signal on the film was collected and immersed in TMbuffer (10 mM Tris-HCl (pH 8.0), 10 mM MgSO₄) to extract the recombinantphages.

With respect to the thus obtained recombinant phages, screening wasrepeated in quite the same manner as described above to obtain 9 singleplaques (positive clones) expected to contain the DNA fragmentconsidered to code for the human-derived tumor cell growth inhibitor.

4) Analysis of nucleotide sequence

From the plaques prepared in 3) described above, DNA was extracted in aconventional manner and its nucleotide sequence was determined by thedideoxy chain terminator method (Sanger, F. et al., Proc. Natl. Acad.Sci. U.S.A., 74, 5463 (1977)), using 7-Deaza-Sequencing Kit (Toyobo Co.,Ltd.). Homology of the nucleotide sequence to the mTG-700 cDNA fragmentwas examined. The results are shown in FIG. 3. As is noted from FIG. 3,comparison reveals that the region similar to a part of the CDNAfragment of mTG-700 precursor was observed from 550 to 675 bases in thenucleotide sequence, cf. FIG. 3 (b) in which the underlined portionindicates a part of the mature mTG-700 cDNA fragment. The region wastranslated into amino acids, which were similar to a part of the mTG-700precursor, see FIG. 3 (c). According to the Cech's rule (Cech, T.D.,Cell, 34, 713, 1983), it is assumed that the 552-675 region would be atranslation site (exon) into a protein, see FIG. 3 (a).

Based on the foregoing results, it is considered that a part of the DNAfragment encoding hTG-700 would be contained in the chromosomal DNAfragment obtained from the plaques prepared in 3) above.

2. Isolation of the cDNA fragment encoding a full-length hTG-700 anddetermination of nucleotide sequence

1) Construction of cDNA library of HCT-15 cells (1) Extraction of mRNAfrom HCT-15 cells Human colonel tumor cells HCT-15 (DainipponPharmaceutical Co., Ltd., Cancer Research, 39, 1020, 1979) were culturedat 37° C. in 10% calf fetal serum-containing F medium in 5% CO₂. At thetime when the cells reached confluence, the medium was removed. Afterwashing once with PBS (-), PBS (-) was supplemented. The cells were thenscraped out with a cell scraper and collected in a conical tube. Aftercentrifugation at 1500×g for 5 minutes at room temperature, PBS (-) wasadded to suspend the cells therein. The suspension was again centrifugedto obtain the precipitates. From the precipitates, MRNA was extractedusing mRNA Extraction Kit (manufactured by Pharmacia). Following thisprocedure, 58.3 μg of mRNA was purified from 2×108 cells.

(2) Synthesis of cDNA

Using the mRNA prepared in (1) as a template, cDNA was synthesized usingoligo dT as a primer, by the use of cDNA Synthesis Kit (manufactured byPharmacia). Following this procedure, 1.0 μg of cDNA was synthesizedfrom 1.9 μg of mRNA.

(4) Ligation of cDNA to vector and in vitro packaging

The cDNA fragment synthesized as described in

(2) above was ligated to EcoRI adapter by the procedure described in1-2)-(2). The in vitro packaging was performed as in the proceduredescribed in 1-2)-(3) to construct cDNA library.

2) Screening of cDNA library of HCT-15 cells The PstI fragment (520 to674 base pairs; see FIG. 3) of the DNA fragment considered to encode apart of hTG-700 obtained in 1-3) described above was labeled with α-32p!dCTP using multi-prime DNA labeling system (Amersham Inc.) to prepare aDNA probe.

Using the DNA probe, screening of the CDNA library for HCT-15 cellsobtained in 1)-(3) described above was performed. The screeningproceeded as in the procedure described in 1-3) above. One positiveclone considered to contain the DNA fragment encoding hTG-700 wasobtained from about 6×10⁵ recombinant phages.

3) Analysis of nucleotide sequence

From the clone obtained in 2) described above, the DNA fragment wasextracted and its nucleotide sequence was determined by the methoddescribed in 1-4) above. FIG. 4 shows a part of the nucleotide sequenceand the amino acid sequence deduced therefrom. Comparison was madebetween the amino acid sequence of hTG-700 expected from the DNAfragment and that of mTG-700, see FIG. 5. As is noted from FIG. 5,comparison reveals high homology in amino acid sequence of hTG-700 tomTG-700. Based on the structure of the N and C termini of mTG-700, itwas assumed that hTG-700 would have Val at the N terminus and Leu at theC terminus (FIG. 4, the underlined portion) and have the amino acidsequence shown in FIG. 6. It was also assumed that the DNA fragmentencoding hTG-700 would have the nucleotide sequence shown in FIG. 6.

EXAMPLE 2 Chemical synthesis of hTG-700

The peptide deduced from the amino acid sequence encoded by thenucleotide sequence shown in FIG. 6 was chemically synthesized by the9-fluorenyl-methoxycarbonyl (Fmoc) method.

The Fmoc method (E. Atherton, C. J. Logan and R. C. Sheppard, J. Chem.Soc., Perkin Trans. 1, 1981, 538-546) is a very simple peptide synthesisfor preparing a linear peptide which comprises repeating a series ofoperations for selective removal of α-amino-protecting Fmoc group fromthe carboxyl terminus of the desired peptide using a secondary amine andcondensation of an amino acid with protected functional groups on theside chain, cutting the thus obtained peptide chain out of the solidphase resin and removing the protective groups on the side chain.

Then, the hTG-700 was synthesized in a 0.1 mmol scale using a peptideautomatic synthesizer (Pepsyn 9050 Peptide Synthesizer: Millipore).Isolation from the solid phase resin with a half amount of the resinafter the synthesis followed by removal of the protective groups on theside chain gave the linear peptide. Since hTG-700 was considered to have3 pairs of S-S bond in the molecule thereof, only the side chain ofcysteine was protected with such a protective group that was not splitoff by a conventional method for removal of protective groups. The thusobtained linear peptide was purified by chemoselective one-steppurification (S. Funakoshi, H. Fukuda and N. Fujii, Proc. Natl. Acad.USA, 88, 6981-6985, 1991) and the protective group for cysteine on theside chain was then removed. Subsequently, air oxidation was performedto form intramolecular S-S bond. The peptide was purified by highperformance liquid chromatography until a single product was obtained.The purified product was analyzed by Protein Sequencer Model 473A(Applied Biosystems); as the result, it was confirmed that the peptidehad the amino acid sequence shown in FIG. 6. In the thus obtainedhTG-700, the S--S bond was formed in the cysteine residues between 6 and19, 14 and 30, and 32 and 41.

Experiment 1

Major cell growth inhibitory activity of hTG-700

With respect to hTG-700 obtained by chemical synthesis in Example 2, theactivity was examined by the following evaluation system.

(1) Growth inhibitory activity on human uterocervical carcinoma HeLa

HeLa cells were inoculated on a 48-well plate (SUMILON) in 2×10⁴ /250μl/10% FBS +DF/well followed by incubation for 24 hours. After themedium was removed, 250 μl of hTG-700-containing DF medium supplementedwith 0.01% BSA was added to the system followed by incubation for 6days. After the incubation, the cells were stripped out with 0.25%trypsin/0.002% EDTA and counted with Coulter Counter (Coulter). Thegrowth inhibitory activity on HeLa cells is shown in terms of % when theactivity in the intact group was made 100%.

The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Growth inhibitory activity on human                                           uterocervical carcinoma HeLa                                                               Cell Count                                                                           (%) of Control                                            ______________________________________                                        Control        2.61 × 10.sup.5                                                                  100                                                   Treated group  0.47 × 10.sup.5                                                                  18                                                    ______________________________________                                    

(2) Growth inhibitory activity on various tumor cells

Tumor cells of HeLa (human uterus cancer), HCT-15 (human colonel tumor),T-13 (human renal tumor) and T-28 (human renal tumor) were inoculated,respectively, on a 48-well plate (SUMILON) in 5×10³ /250 μl /10% FBS+DF/well followed by incubation for 24 hours. After the medium wasremoved, 200 μl of hTG-700-containing DF medium supplemented with 0.1%BSA was added to the system followed by incubation for 6 days. Themedium was removed. After washing with PBS (-), the cells were strippedout with 0.25% trypsin and counted with Coulter Counter (Coulter). Thegrowth inhibitory activity was determined according to the followingequation: ##EQU1##

                  TABLE 2                                                         ______________________________________                                        Growth inhibitory activity on tumor cells                                     hTG-700 (μg/ml)                                                            Cell        10    2          0.4  0.08                                        ______________________________________                                        HeLa        12    47         64   96                                          HCT-15      7     25         38   50                                          T-13        2     8          19   56                                          T-28        4     26         42   75                                          ______________________________________                                    

As is clearly seen from the results of Tables 1 and 2, novel hTG-700provided by the present invention exhibits a tumor cell growthinhibitory activity and is thus expected to be a novel therapeutic agentfor the treatment of tumors.

EXAMPLE 3 Production of hTG-700 by recombinant DNA technology

1. Cloning of DNA fragment encoding hTG-700

(1) Synthesis of oligonucleotides for amplification of hTG-700 gene byPCR

Based on the nucleotide sequence of cDNA of hTG-700, oligonucleotidesused as the primers at the 5' terminus and 3' terminus were prepared,respectively.

That is, as shown in FIG. 7, synthetic oligonucleotide BB-15 containingNcoI linker, a part of the C-terminal coding region of the signalpeptide for MWP (major cell wall protein) gene from Bacillus brevis 47and the region encoding the amino acid in the N-terminal domain ofhTG-700 was synthesized as the 5' primer. As the 3' primer, syntheticoligonucleotide BB-22 was designed and synthesized to have a nucleotidesequence complementary to the DNA sequence containing the C-terminalamino acid-coding region of hTG-700, a termination codon and HindIIIlinker. These oligo-nucleotides were prepared by DNA Synthesizer (ABIInc., Model 380B) and purified by OPC Column (ABI Inc.).

(2) Amplification of hTG-700 gene by PCR and purification

And 58 Al of sterilized water to 10 μl of XgtlO library obtained inExample 1, 2-1)-(3) by cloning cDNA of HCT-15 cells;

Heat the mixture at 95° C. for 10 minutes;

Quench the mixture in ice water;

Add 100 pmols of BB-15, 100 pmols of BB-22,10 μl of x 10 buffer andfinally 16 μl of 1.25 mM dNTP;

Add sterilized water to make the whole volume 100 μl; and,

Add 1 μl of Taq polymerase. Gene amplification by PCR:

Heat at 94° C. for a minute, at 40° C. for 2 minutes and at 72° C. for 3minutes (repeat this cycle 30 times);

Add 10 μl of 3 M sodium acetate (pH 6.0) and 250 μl of ethanol;

Incubate at -70° C. for an hour;

Centrifuge at 10,000×g for 20 minutes;

Precipitate;

Add 300 pl of 70% ethanol;

Centrifuge at 10,000×g for 20 minutes;

Precipitate;

Dry the precipitates by a centrifugal evaporator;

Dissolve in 20 gl of TE buffer;

Add 5 μl of NcoI (12 units/gl), 4 μl of ×10 M buffer (100 mM Tris-HCl(pH 7.5), 100 mM MgCl₂ , 10 mM dithiothreitol, 500 niM NaCl);

Add 11 μl of sterilized water;

Heat at 37° C. for 3 hours;

Heat at 65° C. for 10 minutes for inactivation;

Add 5 μl of HindIII (12 units/μl), 5 μl of ×10 K buffer (200 mM Tris-HCl(pH 8.5), 100 mM MgCl₂₁ , 10 mM dithiothreitol, 1 M KCl );

Heat at 37° C. for 3 hours;

Subject to electrophoresis, 3% NuSieve: agarose (FMC Inc.) =3:1;

Adsorb the amplified DNA fragment onto DE81 Paper (Whatman);

300 μof eluent (1.5 M NaCl, 1 mM EDTA, 10

mM Tris-HCl (pH 8.0))

300 μof phenol:chloroform (1:1)

Agitate the mixture and then centrifuge the same at 10,000×g for 10minutes;

And 750 μof ethanol to the supernatant;

Incubate at -70° C. for an hour;

Centrifuge at 10,000×g for 10 minutes; and,

Dissolve the precipitates in 12 μl of TE buffer (1 mM EDTA, 10 mMTris-HCl (pH 8.0)).

By performing the procedures above, MWP-hTG-700 DNA fragment isobtained, see FIG. 10.

2. Integration of hTG-700 gene in expression plasmid

(1) Construction of pBR-AN

In order to construct pKN120 later described, plasmid pBR-AN having NcoIsite, which will be used for transduction of MWP-hTG-700 bearing a NcoIlinker was constructed. As shown in FIG. 8, PBR-AN was constructed byligating the following two DNAs. Domain A: Two syntheticoligonucleotides BB-1 and Bb-2 were synthesized. After purifying on OPCcolumn, annealing was performed. ##STR4##

Domain A contains the NcoI site and the DNA fragment deleted of MWPsignal peptide-encoding 5' terminal region. Domain B: BamHI-NruIfragment from E. coli plasmid pBR322 (Sutchliffe, J. G., Cold SpringHarbor Symposium, 43, 77-90, 1979)

(2) Digestion of M13mp18RFDNA by SmaI-HindIII and dephosphorylation

After 2 μg of M13mpl8RFDNA (Takara Shuzo) was digested by 24 units eachof SmaI and HindIII at 37° C. for 3 hours, the digestion product wasthen dephosphorylated with calf intestinal phosphatase (Takara Shuzo).The product was dissolved in TE buffer in a final concentration of 0.1μg/μl to obtain SmaI-HindIII DNA fragment of M13mpl8RF, see FIG. 9.

(3) Preparation of cloning vector pKN120

Ten micrograms of plasmid pBR-AN bearing a DNA sequence encoding theNcoI site and a part of the C-terminal region of the signal peptide forMAP (hereinafter referred to as NcoI-MWP region) was digested by 100units of HindIII and 100 units of PvuII at 37° C. for 2 hours. Thereaction product was electrophoresed on 3% NuSieve:agarose (3:1). Thenthe HindIII-ApaLl fragment containing the NcoI-MWP region was isolatedand purified using DE81 paper and finally dissolved in 12 μl of TEbuffer.

The resulting DNA solution (5 μl) was mixed with 2 μl of theSmaI-HindIII DNA fragment prepared in (2) above to effect ligrationusing T4 DNA ligase (Takara Shuzo). The reaction solution was transducedinto E. coli JM105 (C. Yanisch-Perron, J. Vieira and J. Messing, Gene33, 103-119, 1985) by the calcium method (Messing, J., Methods inEnzymology, 101, 20-78, 1983) to form plaques using JM105 as anindicator. The plaques were adsorbed to JM105 and incubated. From thecells RF-DNA was prepared by the alkali method (Birnboim, H. C. andDoly, J., Nucleic Acids Res., 7, 1513-1523, 1929). The desired plasmid,pKNl20, bearing the HindIII-ApaLI DNA fragment containing the NcoI-MWPregion was thus obtained, see FIG. 9.

(4) Cloninq of hTG-700 gene to pKN120

After 2 μg of pKN120 was digested by NcoI-HindIII, the digestion productwas dephosphorylated with calf intestinal phosphatease (Takara Shuzo).The product was dissolved in TE buffer in a final concentration of 0.1μg/pl, see FIG. 10. The resulting DNA solution (2 μl) was mixed with 5μl of a DNA solution of MWP-hTG-700 containing hTG-700 amplified in1-(2) above to effect ligation using T4 DNA ligase. The reactionsolution was transformed to E. coli JM105 by the calcium method. RFDNAwas prepared from the transformant according to the alkali method toobtain plasmid 46hTG-RF in which the hTG-700 gene was ligated downstreamof the MWP signal peptide-coding region, see FIG. 11.

(5) Construction of expression plasmid PBB230

1) After 5 μg of pNU200 was digested by 50 units of ApaLI and 50 unitsof HindIII at 37° C. for 3 hours, the digestion product wasdephosphorylated by calf intestinal phosphatase. After electrophoresison agarose gel, vector DNA containing the promoter of MWP and SDsequence, the 5' terminal region of the DNA nucleotide sequence encodingsignal peptide and erythromycin-resistant gene was purified using DE81paper. The purified vector DNA was dissolved in 30 μl of TE buffer.

2) After 20 pg of 46hTG-RF was digested by 50 units each of restrictionenzymes ApaLI and HindIII at 37° C. for 3 hours, the digestion productwas subjected to electrophoresis on agarose gel. Thereafter, theApaLI-HindIII DNA fragment was isolated and purified using DE81 paper toobtain the DNA fragment ligated with hTG-700 gene downstream of the DNAsequence encoding the 5' terminus-deleted MWP signal peptide, see FIG.11. The DNA fragment was dissolved in 12 μl of TE buffer.

3) Ligation was performed between 2 μl of the dephosphorylated vectorprepared in 1) above and 5 μl of the inserted DNA fragment containinghTG-700 gene, using T4 DNA ligase. These reaction solutions weretransformed to Bacillus brevis 47-5 (H. Yamagata et al., J. Bacteriol.,169, 1239-1245, 1987) by the method of Takahashi et al., J. Bacteriol.,156, 1130-1134, 1983). Plasmid DNA was prepared from the transformant bythe alkali method. Thus, pBB230 which was an expression plasmid of thedesired hTG-700 and ligated to MWP promoter, SD sequence and hTG-700gene downstream of the signal peptide-coding region, see FIG. 11.

3. Production of hTG-700 in Bacillus brevis

Bacillus brevis bearing pBB230 (pBB230/47-5) prepared in 2-(5) above wasshake-cultured at 30° C. for 3 days in an appropriate medium, forexample, T₃ modified medium (5 mM MgCl₂, 20 g Polypeptone P1 (NipponPharmaceutical Co., Ltd.), 6.5 g yeast extract (Nippon PharmaceuticalCo., Ltd.), 30 g glucose, 0.1 g uracil, per litter, adjusted pH to 7.0with NaOH) supplemented with 10 μg/ml of erythromycin. Thereafter 20 g/lof glucose was added thereto followed by shake culture for further 2days.

4. Assay for biological activity of hTG-700 produced by Bacillus brevis

For the purpose of assaying for biological activity, the culturesupernatant was centrifuged and sterilized through ULTRAFREE C3 HV STRL(Millipore). The biological activity of hTG-700 in the supernatant wasdetermined by growth stimulation activity on mouse Balb3T3 cells.

Mouse fibroblast-derived Balb3T3 was inoculated on 5% calfserum-containing Dulbecco's modified Eagle's medium charged in a 96-wellplate in an amount of 1×10⁴ /100 μl medium/well. Incubation wasperformed at 37° C. for 48 hours under 5% CO₂ conditions. The culturebroth was then changed by 0.5% calf serum-containing Dulbecco's modifiedEagle's medium (0.5% CS-DME), followed by incubation for further 24hours. The medium was then changed with 0.5% CS-DME containing 0.1 μg/mlto 100 ng/ml of authentic hTG-700 or a sample, followed by incubation at37°C. for 20 hours. Labeling was effected for 4 hours by adding 0.25μCi/well of ³ H! thymidine (Amersham) and ³ H! thymidine intake activitywas quantitatively determined by β-plate system (Amersham). As theresult, about 4 mg of hTG-700 was produced per liter of the medium.

5. Purification of hTG-700 produced by Bacillus brevis

(1) O-Sepharose-C4. resin column

After 100 liters of the culture supernatant of pBB230/47-5 incubated inthe manner shown in 3. above was separated with S type ultracentrifuging machine (8,000 rpm, Kokusan Seiko), the supernatant wascollected and filtered through Peristaltic Pump Cassette System(ultrafiltering membrane system, 0.3 μm filter, Fuji Filter). Thefiltrate was treated with 1 M Tris-HCl (pH 7.5) to adjust pH to 7.5 andwas added to Q-Sepharose-C₄ resin column, which has been previouslyequilibrated with 5% acetonitrile.

    ______________________________________                                        Column:       Q Sepharose FF (Pharmacia)                                                    φ16 cm × 10 cm                                                      C.sub.4 resin (Chemco), φ16 cm × 15 cm                Flow rate:    40 ml/min                                                       ______________________________________                                    

hTG-700 is non-adsorptive to Q Sepharose but adsorbed to C₄ resin. Afteradsorption, the C₄ column was washed with 5% acetonitrile and elutedwith 50% acetonitrile and 0.1% TFA solution (flow rate: 40 ml/min).

(2) S-Sepharose

Acetonitrile was removed from the active fraction obtained in (1) above,using an evaporator and 1 M acetate buffer (pH 4.0, sodiumacetate-acetic acid) was then added to adjust the pH of the sample to 4.The sample was adsorbed to S-Sepharose column which had beenequilibrated with 20 mYM acetate buffer and 5% acetonitrile. Elution wasstepwise performed with eluent A and eluent B.

    ______________________________________                                        Column:       S-Sepharose FF (Pharmacia)                                                    φ10 cm × 10 cm                                        Eluent A:     20 mM acetate buffer (pH 4.0), 5%                                             acetonitrile + 0.05 M NaCl                                      Eluent B:     20 mM acetate buffer (pH 4.0), 5%                                             acetonitrile + 0.5 M NaCl                                       Flow rate:    50 ml/min                                                       ______________________________________                                    

(3) C₄ HPLC

The active fraction was obtained from those eluted with eluent B andpurified by C₄ reverse phase HPLC.

    ______________________________________                                        Column:         C.sub.4, φ2.2 cm × 25 cm (Vydac)                    Eluent A:       10% acetonitrile, 0.1% TFA                                    Eluent B:       40% acetonitrile, 0.1% TFA                                    Flow rate:      15 ml/min                                                     Fractionation:  2 min                                                         Gradient:       A → B (150 min)                                        ______________________________________                                    

(4) C₁₈ HPLC

After an equal volume of 0.1% TFA solution was added to the activefraction (3) above, separation and purification were performed throughC₁₈ column.

    ______________________________________                                        Column:    μBondsphere C.sub.18 (φ39 cm × 150 mm, Waters         Eluent A:  0.1% TFA                                                           Eluent B:  20% acetonitrile, 0.1% TFA                                         Eluent C:  40% acetonitrile, 0.1% TFA                                         Flow rate: 8 ml/min                                                           Conditions:                                                                              A → B (5 mins) → B (5 mins) → C (120                     mins)                                                              ______________________________________                                    

As the result, the activity was focused on a single peak, see FIG. 12.

Experiment 2

(1) Tumor cell growth inhibitory activity of hTG-700

Tumor cells from HeLa (human uterus cancer), HCT-15 (human colon tumor),T-13 (human renal tumor) and T-28 (human renal tumor) cells wereinoculated on each well of a 48 well plate in an amount of 5×10³ /150 μlof 10% calf fetal serum-supplemented DF culture broth (Dulbecco'smodified MEM:HamF-12=1:1). After incubation for 24 hours, the medium wasremoved and 200 μl of 0.1% BAS-supplemented DF culture broth containinghTG-700, which had been obtained in Example 3, 5 above, was added to thecells followed by incubation for 6 days. The culture broth was thenremoved. After washing with PBS(-) (0.2 g KC1, 0.2 g KH₂ PO₄ , 8 g NaCl,1.15 g Na₂ HPO₄, per litter), the pellet was stripped out with 0.25%trypsin and the cell count was counted with Coulter Counter (Coulter).The cell growth inhibitory activity was determined according to thefollowing equation: ##EQU2##

As is appreciated from FIG. 13, the cell growth inhibitory activity ofhTG-700 was observed with respect to some of human tumor cells.

(2) Detection of hTG-700 by mouse G-700 antibody

After 20% SDS-PAGE was performed on hTG-700 obtained in Example 3, 5above, hTG-700 was transferred onto a nitrocellulose filter. Aftershaking at room temperature for an hour with 2% bovine serum albumin(BSA)-supplemented TBS solution (150 mnM NaCl, 20 mM pH 8.0), the filterwas immersed in 0.1% BSA-Tris-HCl, supplemented TBS solution and allowedto stand at 4° C. overnight followed by blocking. The reaction wassequentially carried out using anti-mouse TG-700 antibody and donkeyanti-rabbit IgGF (ab') ₂ as primary and secondary antibodies,respectively. Washing between the reactions was carried out with TBSsolution containing 2% Tween 20 and 10% Block Ace (DainipponPharmaceutical Co., Ltd.). Using ECL System (Amersham Inc.), hTG-700 wasdetected. Human type TG-700 was detected by the antibody to mouse TG-700at the position of the expected molecular weight, see FIG. 14.Industrial Applicability

The present invention provides novel hTG-700. This hTG-700 is expectedto be effective for the treatment of leukemia, renal cancer anduterocervical cancer. The present invention further provides the DNAfragment encoding hTG-700. Utilizing the DNA fragment, hTG-700 can beproduced by recombinant DNA technique. According to the presentinvention, hTG-700 can be expressed in Bacillus brevis, using thepromoter and the DNA fragment encoding signal peptide as a regulatorgene, originating from Bacillus brevis, whereby hTG-700 can be secretedout of the cell in such a form that the active steric structure ismaintained. Therefore, the present invention provides hTG-700 which isexpected as a new drug also provides an industrially advantageous methodfor producing hTG-700.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ValSerIleThrLysCysSerSerAspMetAsnGlyTyrCysLeuHis                              151015                                                                        GlyGlnCysIleTyrLeuValAspMetSerGlnAsnTyrCysArgCys                              202530                                                                        GluValGlyTyrThrGlyValArgCysGluHisPhePheLeu                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTGTCAATAACAAAGTGTAGCTCTGACATGAATGGCTATTGTTTGCATGGACAGTGCATC60                TATCTGGTGGACATGAGTCAAAACTACTGCAGGTGTGAAGTGGGTTATACTGGTGTCCGA120               TGTGAACACTTCTTTTTA138                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGTGCACTCGCACTTACTGTTGCTCCCATGGCTTTCGCTGCAG43                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GATCCTGCAGCGAAAGCCATGGGAGCAACAGTAAGTGCGAGTGCACT47                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..138                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTGCAGATTACAAAGTGTAGTTCTGACATGGACGGCTACTGCTTGCAT48                            ValGlnIleThrLysCysSerSerAspMetAspGlyTyrCysLeuHis                              151015                                                                        GGCCAGTGCATCTACCTGGTGGACATGAGAGAGAAATTCTGCAGATGT96                            GlyGlnCysIleTyrLeuValAspMetArgGluLysPheCysArgCys                              202530                                                                        GAAGTGGGCTACACTGGTCTGCGATGTGAGCACTTCTTTCTA138                                 GluValGlyTyrThrGlyLeuArgCysGluHisPhePheLeu                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ValGlnIleThrLysCysSerSerAspMetAspGlyTyrCysLeuHis                              151015                                                                        GlyGlnCysIleTyrLeuValAspMetArgGluLysPheCysArgCys                              202530                                                                        GluValGlyTyrThrGlyLeuArgCysGluHisPhePheLeu                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CAGTTCAGACAGAAGACAATCCACGTGTGGCTCAAGTGTCAATAACAAAGTGTAGCTCTG60                ACATGAATGGCTATTGTTTGCATGGACAGTGCATCTATCTGGTGGACATGAGTCAAAACT120               ACTGCAGGT129                                                                  (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TAGTTCAGATGGAAGACGATCCCCGTGTGGCTCAAGTGCAGATTACAAAGTGTAGTTCTG60                ACATGGACGGCTACTGCTTGCATGGCCAGTGCATCTACCTGGTGGACATGAGAGAGAAAT120               TCTGCAGAT129                                                                  (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..120                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CAGACAGAAGACAATCCACGTGTGGCTCAAGTGTCAATAACAAAGTGT48                            GlnThrGluAspAsnProArgValAlaGlnValSerIleThrLysCys                              151015                                                                        AGCTCTGACATGAATGGCTATTGTTTGCATGGACAGTGCATCTATCTG96                            SerSerAspMetAsnGlyTyrCysLeuHisGlyGlnCysIleTyrLeu                              202530                                                                        GTGGACATGAGTCAAAACTACTGC120                                                   ValAspMetSerGlnAsnTyrCys                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlnThrGluAspAsnProArgValAlaGlnValSerIleThrLysCys                              151015                                                                        SerSerAspMetAsnGlyTyrCysLeuHisGlyGlnCysIleTyrLeu                              202530                                                                        ValAspMetSerGlnAsnTyrCys                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..120                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CAGATGGAAGACGATCCCCGTGTGGCTCAAGTGCAGATTACAAAGTGT48                            GlnMetGluAspAspProArgValAlaGlnValGlnIleThrLysCys                              151015                                                                        AGTTCTGACATGGACGGCTACTGCTTGCATGGCCAGTGCATCTACCTG96                            SerSerAspMetAspGlyTyrCysLeuHisGlyGlnCysIleTyrLeu                              202530                                                                        GTGGACATGAGAGAGAAATTCTGC120                                                   ValAspMetArgGluLysPheCys                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GlnMetGluAspAspProArgValAlaGlnValGlnIleThrLysCys                              151015                                                                        SerSerAspMetAspGlyTyrCysLeuHisGlyGlnCysIleTyrLeu                              202530                                                                        ValAspMetArgGluLysPheCys                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 433 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..368                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TCACAGCTTTAGTTCAGACAGAAGACAATCCACGTGTGGCTCAAGTG47                             ThrAlaLeuValGlnThrGluAspAsnProArgValAlaGlnVal                                 151015                                                                        TCAATAACAAAGTGTAGCTCTGACATGAATGGCTATTGTTTGCATGGA95                            SerIleThrLysCysSerSerAspMetAsnGlyTyrCysLeuHisGly                              202530                                                                        CAGTGCATCTATCTGGTGGACATGAGTCAAAACTACTGCAGGTGTGAA143                           GlnCysIleTyrLeuValAspMetSerGlnAsnTyrCysArgCysGlu                              354045                                                                        GTGGGTTATACTGGTGTCCGATGTGAACACTTCTTTTTAACCGTCCAC191                           ValGlyTyrThrGlyValArgCysGluHisPhePheLeuThrValHis                              505560                                                                        CAACCTTTAAGCAAAGAGTATGTGGCTTTGACCGTGATTCTTATTATT239                           GlnProLeuSerLysGluTyrValAlaLeuThrValIleLeuIleIle                              657075                                                                        TTGTTTCTTATCACAGTCGTCGGTTCCACATATTATTTCTGCAGATGG287                           LeuPheLeuIleThrValValGlySerThrTyrTyrPheCysArgTrp                              80859095                                                                      TACAGAAATCGAAAAAGTAAAGAACCAAAGAAGGAATATGAGAGAGTT335                           TyrArgAsnArgLysSerLysGluProLysLysGluTyrGluArgVal                              100105110                                                                     ACCTCAGGGGATCCAGAGTTGCCGCAAGTCTGAATGGCGCCATCAAACTT385                         ThrSerGlyAspProGluLeuProGlnVal                                                115120                                                                        ATGGGCCAGGGATAACAGTGTGCCTGGTTAATATTAATATTCCATTTT433                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ThrAlaLeuValGlnThrGluAspAsnProArgValAlaGlnValSer                              151015                                                                        IleThrLysCysSerSerAspMetAsnGlyTyrCysLeuHisGlyGln                              202530                                                                        CysIleTyrLeuValAspMetSerGlnAsnTyrCysArgCysGluVal                              354045                                                                        GlyTyrThrGlyValArgCysGluHisPhePheLeuThrValHisGln                              505560                                                                        ProLeuSerLysGluTyrValAlaLeuThrValIleLeuIleIleLeu                              65707580                                                                      PheLeuIleThrValValGlySerThrTyrTyrPheCysArgTrpTyr                              859095                                                                        ArgAsnArgLysSerLysGluProLysLysGluTyrGluArgValThr                              100105110                                                                     SerGlyAspProGluLeuProGlnVal                                                   115120                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ValGlnIleThrLysCysSerSerAspMetAspGlyTyrCysLeuHis                              151015                                                                        GlyGlnCysIleTyrLeuValAspMetArgGluLysPheCysArgCys                              202530                                                                        GluValGlyTyrThrGlyLeuArgCysGluHisPhePheLeu                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ValSerIleThrLysCysSerSerAspMetAsnGlyTyrCysLeuHis                              151015                                                                        GlyGlnCysIleTyrLeuValAspMetSerGlnAsnTyrCysArgCys                              202530                                                                        GluValGlyTyrThrGlyValArgCysGluHisPhePheLeu                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..138                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GTGTCAATAACAAAGTGTAGCTCTGACATGAATGGCTATTGTTTGCAT48                            ValSerIleThrLysCysSerSerAspMetAsnGlyTyrCysLeuHis                              151015                                                                        GGACAGTGCATCTATCTGGTGGACATGAGTCAAAACTACTGCAGGTGT96                            GlyGlnCysIleTyrLeuValAspMetSerGlnAsnTyrCysArgCys                              202530                                                                        GAAGTGGGTTATACTGGTGTCCGATGTGAACACTTCTTTTTA138                                 GluValGlyTyrThrGlyValArgCysGluHisPhePheLeu                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ValSerIleThrLysCysSerSerAspMetAsnGlyTyrCysLeuHis                              151015                                                                        GlyGlnCysIleTyrLeuValAspMetSerGlnAsnTyrCysArgCys                              202530                                                                        GluValGlyTyrThrGlyValArgCysGluHisPhePheLeu                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..30                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CCCATGGCTTTCGCTGTGTCAATAACAAAG30                                              MetAlaPheAlaValSerIleThrLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      MetAlaPheAlaValSerIleThrLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CAAGCTTTTATTATAAAAAGAAGTGTTCACA31                                             __________________________________________________________________________

We claim:
 1. An isolated human-derived tumor cell growth inhibitorconsisting of the amino acid sequence shown by formula (1) (Seq. I.D.No. 1): ##STR5##
 2. An isolated DNA fragment encoding a human tumor cellgrowth inhibitor according to a claim
 1. 3. An isolated DNA fragmentaccording to claim 2, which consists of the nucleotide sequence shown byformula (2) (Seq. I.D. No. 2): ##STR6##
 4. A DNA sequence comprising apromoter originating from Bacillus brevis, a DNA fragment encoding asignal peptide originating from Bacillus brevis and a DNA fragmentaccording to claims
 3. 5. An expression plasmid comprising a DNAsequence according to claim
 4. 6. Bacillus brevis transformed by anexpression plasmid according to claim
 5. 7. A method for producing ahuman-derived tumor cell growth inhibitor by genetic engineering whichcomprises culturing Bacillus brevis of claim 6 under conditions wherebysaid human-derived tumor cell growth inhibitor is expressed and secretedextra cellularly, and then recovering said human-derived tumor cellgrowth inhibitor from the culture broth.